This proposal will fund genetic, structural, and mechanistic studies of prenyltransferases, essential enzymes in the biosynthesis of cholesterol, prenylated proteins, and a large number of other essential secondary metabolites. High levels of serum cholesterol are a major factor in coronary heart disease, the leading cause of death in the U.S. Familial hypercholesterolemia, a genetic predisposition to high blood cholesterol that occurs in 0.2% of the human population, increases the risk of death from heart disease by 20-fold, and requires active long-term intervention with drugs for treatment. A substantial number of cancers, the second leading cause of death in the U.S., are caused by mutations that convert H-ras, K-ras, and N-ras into active oncogenies. These include cancers of the pancreas (90%), colon (50%), thyroid (50%), and lung (30%), as well as myeloid leukemia (30%) and melanoma (20%). The oncogenic activity of Ras proteins requires that they associate with the inner surface of the outer membrane, and this association requires a posttranslational modification of the proteins by a farnesyl moiety. Related posttranslational modifications have been implicated in chloroderemia, a genetic disorder that leads to retinal degeneration, and infection by the virulent hepatitis delta virus. This proposal outlines a study of prenyltransferases, a family of enzymes that catalyze the major reactions in the trunk of the isoprenoid pathway and all of the branch-point reactions leading to over 23,000 known metabolites. Several of these enzymes are logical therapeutic targets. Farnesyl diphosphate synthase, geranylgeranyl diphosphate synthase, hexaprenyl diphosphate synthase, protein farnesyl transferase, protein geranylgeranyl transferase (types I and II), AMP dimethylallyltransferase, tRNA dimethylallyl transferase, geranylgeranylglyceryl phosphate synthase, and dimethylallyltryptophan synthase will be studied. The proteins comprise a diverse phylogenetic and structural group that catalyzes alkylation of electron-rich olefinic, aromatic, and heteroatom enters by allylic isoprenoid diphosphates. We plan to establish the chemical and binding mechanisms for different prenyltransferases, to identify structural motifs responsible for binding and catalysis, and to develop potent new inhibitors based on structural and mechanistic concepts. To accomplish this task we will continue our efforts to i) identify and characterize genes in the isoprenoid pathway ii) develop bacterial and eucaryal strains for overproduction of isoprenoid enzymes, iii) measure the steady-state and pre-steady-state rates, iv) define the chemical mechanisms of the reactions, v) synthesize and evaluate new inhibitors, i) obtain structural information by X-ray and NMR studies, vii) identify important active-site residues by affinity labeling and site-directed mutagenesis, and viii) develop new tools to facilitate these studies.